基于实时荧光聚合酶链式反应快速检测食源性大肠埃希氏菌O157:H

Rapid detection of food-borne Escherichia coli O157:H7 based on real-time fluorescence polymerase chain reaction

目的 建立一种快速、灵敏、特异、高效的食源性大肠埃希氏菌O157:H7型实时荧光聚合酶链式反应(polymerase chain reaction,PCR)检测方法。方法 针对大肠埃希氏菌O157:H7型的O抗原特异基因rfbE保守区域设计特异性引物和探针,合成基因片段绘制标准曲线,在菌液基因组DNA和质粒双层面调试优化以完成方法的初步建立。其后,对该方法的特异性、敏感性、重复性等进行全面的质量评估验证。结果 该方法特异性100%;基因组DNA检测敏感性为7.11×10^(2)fg/μL;纯培养物水平检测敏感性为1.0×10^(2)CFU/mL;重复性变异系数在0.10%~1.00%之间;标准曲线相关系数r^(2)为0.9994。结论 成功建立了一种性能良好的大肠埃希氏菌O157:H7型实时荧光探针PCR快速检测方法,该方法具有灵敏度高、特异性强、扩增时间短,仅为35 min的特点,可用于疑似大肠埃希氏菌O157:H7型污染样品的快速诊断检测。

Objective To establish a rapid,sensitive,specific and efficient real-time polymerase chain reaction(PCR) method for the detection of food-borne Escherichia. coli O157:H7. Methods Specific primers and probes were designed for the conserved region of the O antigen-specific gene rfbE of E.coli O157:H7. Standard curves were drawn by synthesizing gene fragments,and double-level debugging and optimization of genomic DNA and plasmid in bacterial solution were performed to complete the preliminary establishment of the method. Then,the specificity,sensitivity and repeatability of the method were evaluated and verified. Results The specificity of this method was 100%,the sensitivity of genomic DNA was 7.11×10^(2) fg/μL,the sensitivity of pure culture level was 1.0×10^(2) CFU/mL,the coefficient of variation of repeatability was between 0.10% and 1.00%,the correlation coefficient r^(2) of the standard curve was 0.9994. Conclusion A good real-time fluorescent probe PCR method for rapid detection of E. coli O157:H7 is successfully established. The method has the characteristics of high sensitivity,strong specificity and short amplification time(only 35 min),and can be used for the rapid diagnostic detection of suspected E. coli O157:H7 contaminated samples.